1887

Abstract

A group B streptococcal (GBS) bacteriophage lysin gene was cloned and expressed in . The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other -haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including . The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27063-0
2004-07-01
2019-08-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502079.html?itemId=/content/journal/micro/10.1099/mic.0.27063-0&mimeType=html&fmt=ahah

References

  1. Anthony, B. F., Okada, D. M. & Hobel, C. J. ( 1978; ). Epidemiology of group B Streptococcus: longitudinal observations during pregnancy. J Infect Dis 137, 524–530.[CrossRef]
    [Google Scholar]
  2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Seidman, J. G., Smith, J. A. & Struhl, K. ( 1998; ). Current Protocols in Molecular Biology, pp. 1.13.1–1.13.4. New York: Wiley.
  3. Bateman, A. & Rawlings, N. D. ( 2003; ). The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases. Trends Biochem Sci 28, 234–237.[CrossRef]
    [Google Scholar]
  4. Centers for Disease Control and Prevention ( 1996; ). Prevention of perinatal group B streptococcal disease: a public health perspective. Morb Mortal Wkly Rep 45 (no. RR-7), 1–24.
    [Google Scholar]
  5. Fischetti, V. A., Gotschlich, E. C. & Bernheimer, A. W. ( 1971; ). Purification and physical properties of group C streptococcal phage-associated lysin. J Exp Med 133, 1105–1117.[CrossRef]
    [Google Scholar]
  6. Grundling, A., Manson, M. D. & Young, R. ( 2001; ). Holins kill without warning. Proc Natl Acad Sci U S A 98, 9348–9352.[CrossRef]
    [Google Scholar]
  7. Haug, R. H., Gudding, R. & Bakken, G. ( 1981; ). Serotyping and bacteriophage typing of human and bovine group-B streptococci. J Med Microbiol 14, 479–482.[CrossRef]
    [Google Scholar]
  8. Hauth, J. C., Macpherson, C., Carey, J. C. & 9 other authors ( 2003; ). Early pregnancy threshold vaginal pH and Gram stain scores predictive of subsequent preterm birth in asymptomatic women. Am J Obstet Gynecol 188, 831–835.[CrossRef]
    [Google Scholar]
  9. Jado, I., Lopez, R., Garcia, E., Fenoll, A., Casal, J. & Garcia, P. ( 2003; ). Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J Antimicrob Chemother 52, 967–973.[CrossRef]
    [Google Scholar]
  10. Karakawa, W. W. & Krause, R. M. ( 1966; ). Studies on the immunochemistry of streptococcal mucopeptide. J Exp Med 124, 155–171.[CrossRef]
    [Google Scholar]
  11. Loeffler, J. M. & Fischetti, V. A. ( 2003; ). Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrob Agents Chemother 47, 375–377.[CrossRef]
    [Google Scholar]
  12. Loeffler, J. M., Nelson, D. & Fischetti, V. A. ( 2001; ). Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294, 2170–2172.[CrossRef]
    [Google Scholar]
  13. Marchler-Bauer, A., Panchenko, A. R., Shoemaker, B. A., Thiessen, P. A., Geer, L. Y. & Bryant, S. H. ( 2002; ). CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res 30, 281–283.[CrossRef]
    [Google Scholar]
  14. Marchler-Bauer, A., Anderson, J. B., DeWeese-Scott, C. & 24 other authors ( 2003; ). CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res 31, 383–387.[CrossRef]
    [Google Scholar]
  15. Mokrasch, L. C. ( 1967; ). Use of 2,4,6-trinitrobenzenesulfonic acid for the coestimation of amines, amino acids and proteins in mixtures. Anal Biochem 18, 64–71.[CrossRef]
    [Google Scholar]
  16. Navarre, W. W., Ton-That, H., Faull, K. F. & Schneewind, O. ( 1999; ). Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a d-alanyl-glycine endopeptidase activity. J Biol Chem 274, 15847–15856.[CrossRef]
    [Google Scholar]
  17. Nelson, D., Loomis, L. & Fischetti, V. A. ( 2001; ). Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Acad Sci U S A 98, 4107–4112.[CrossRef]
    [Google Scholar]
  18. Nelson, D., Schuch, R., Zhu, S., Tscherne, D. M. & Fischetti, V. A. ( 2003; ). Genomic sequence of C1, the first streptococcal phage. J Bacteriol 185, 3325–3332.[CrossRef]
    [Google Scholar]
  19. Patron, R. L., Climo, M. W., Goldstein, B. P. & Archer, G. L. ( 1999; ). Lysostaphin treatment of experimental aortic valve endocarditis caused by a Staphylococcus aureus isolate with reduced susceptibility to vancomycin. Antimicrob Agents Chemother 43, 1754–1755.
    [Google Scholar]
  20. Regan, J. A., Klebanoff, M. A., Nugent, R. P. & 7 other authors ( 1996; ). Colonization with group B streptococci in pregnancy and adverse outcome. Am J Obstet Gynecol 174, 1354–1360.[CrossRef]
    [Google Scholar]
  21. Reinscheid, D. J., Stosser, C., Ehlert, K., Jack, R. W., Moller, K., Eikmanns, B. J. & Chhatwal, G. S. ( 2002; ). Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcus. Microbiology 148, 3245–3254.
    [Google Scholar]
  22. Riordan, J. F. & Vallee, B. L. ( 1967; ). Acetylation. Methods Enzymol 11, 565–570.
    [Google Scholar]
  23. Russell, H., Norcross, N. L. & Kahn, D. E. ( 1969; ). Isolation and characterization of Streptococcus agalactiae bacteriophage. J Gen Virol 5, 315–317.[CrossRef]
    [Google Scholar]
  24. Sanger, F., Nicklen, S. & Coulson, A. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.[CrossRef]
    [Google Scholar]
  25. Schindler, C. A. & Schuhardt, V. T. ( 1964; ). Lysostaphin: a new bacteriolytic agent for the staphylococcus. Proc Natl Acad Sci U S A 51, 414–421.[CrossRef]
    [Google Scholar]
  26. Schleifer, K. H. & Kandler, O. ( 1972; ). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
    [Google Scholar]
  27. Schuch, R., Nelson, D. & Fischetti, V. A. ( 2002; ). A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418, 884–889.[CrossRef]
    [Google Scholar]
  28. Smith, R. F. & Willett, N. P. ( 1968; ). Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms. Appl Microbiol 16, 1434–1436.
    [Google Scholar]
  29. Spiro, R. G. ( 1966; ). Analysis of sugars found in glycoproteins. Methods Enzymol 8, 3–26.
    [Google Scholar]
  30. Stringer, J. ( 1980; ). The development of a phage-typing system for group-B streptococci. J Med Microbiol 13, 133–143.[CrossRef]
    [Google Scholar]
  31. Takahashi, S., Detrick, S., Whiting, A. A., Blaschke-Bonkowksy, A. J., Aoyagi, Y., Adderson, E. E. & Bohnsack, J. F. ( 2002; ). Correlation of phylogenetic lineages of group B streptococci, identified by analysis of restriction-digestion patterns of genomic DNA, with infB alleles and mobile genetic elements. J Infect Dis 186, 1034–1038.[CrossRef]
    [Google Scholar]
  32. Young, R., Wang, I.-N. & Roof, W. D. ( 2000; ). Phages will out: strategies of host cell lysis. Trends Microbiol 8, 120–128.[CrossRef]
    [Google Scholar]
  33. Yow, M. D., Leeds, L. J., Thompson, P. K., Mason, E. O., Jr, Clark, D. J. & Beachler, C. W. ( 1980; ). The natural history of group B streptococcal colonization in the pregnant woman and her offspring. I. Colonization studies. Am J Obstet Gynecol 137, 34–38.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27063-0
Loading
/content/journal/micro/10.1099/mic.0.27063-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error